CDKL5 regulates p62-mediated selective autophagy and confers protection against neurotropic viruses

Virophagy, the selective autophagosomal engulfment and lysosomal degradation of viral components, is crucial for neuronal cell survival and antiviral immunity. However, the mechanisms leading to viral antigen recognition and capture by autophagic machinery remain poorly understood. Here, we identified cyclin-dependent kinase–like 5 (CDKL5), known to function in neurodevelopment, as an essential regulator of virophagy. Loss-of-function mutations in CDKL5 are associated with a severe neurodevelopmental encephalopathy. We found that deletion of CDKL5 or expression of a clinically relevant pathogenic mutant of CDKL5 reduced virophagy of Sindbis virus (SINV), a neurotropic RNA virus, and increased intracellular accumulation of SINV capsid protein aggregates and cellular cytotoxicity. Cdkl5-knockout mice displayed increased viral antigen accumulation and neuronal cell death after SINV infection and enhanced lethality after infection with several neurotropic viruses. Mechanistic studies demonstrated that CDKL5 directly binds the canonical selective autophagy receptor p62 and phosphorylates p62 at T269/S272 to promote its interaction with viral capsid aggregates. We found that CDKL5-mediated phosphorylation of p62 facilitated the formation of large p62 inclusion bodies that captured viral capsids to initiate capsid targeting to autophagic machinery. Overall, these findings identify a cell-autonomous innate immune mechanism for autophagy activation to clear intracellular toxic viral protein aggregates during infection.


Assays for autophagy assessment
To determine the induction of autophagy in HeLa cells and primary cortical neurons (3), the numbers of autophagosomes as represented by GFP-LC3 puncta per cells were counted by an observer blinded to the condition or cell genotype by fluorescence microscopy.In addition, LC3-I to LC3-II conversion was determined by western blot using anti-LC3B antibody.For virophagy experiments, cells were infected with SINV strains MOI of 10 in Opti-MEM I Reduced Serum Medium supplemented with 5% FBS (culture medium) for 1h then exchanged with fresh culture media or maintained in culture media only (mock).For amino-acid starvation, cells were treated for either 1h (GFP-LC3 microscopy assays) or 3 h (LC3 I/II western blots) with Earle's Balanced Salt Solution (EBSS, Thermo Fisher Scientific) at 37℃ and for mTOR inhibition, with 250 nM of torin 1 (Selleck Chemicals S2827) or DMSO as the vehicle control for the same periods of time.
Autophagic flux was assessed by exposing cells to BafA1 (100nM) for 2h.

Generation of dsTE12Q.HA-capsid virus
For the generation of dsTE12Q.HA-capsid virus, a geneblock fragment "CATCTGACTAATACTACAACACCACCACCATGTACCCGTATGATGTTCCGGATTAC GCTGGCTATCCCTACGACGTGCCCGACTATGCCGGGTACCCCTATGACGTCCCAGAC TACGCA AATAGAGGATTCTTTAACATGCTCGGC" containing a partial capsid sequence and the in-frame 3xHA coding sequence was synthesized (IDT).The dsTE12Q recombinant vector was linearized by PCR with primers "AATAGAGGATTCTTTAACATGCTCGGC" and "GGTGGTGGTGTTGTAGTATTAGTCAGATG" immediately after the capsid start codon.The linearized vector and geneblock were then mixed to create the dsTE12Q.HA-Capsid recombinant SINV vector using NEBuilder ® HiFi DNA Assembly Master Mix per manufacturer's instructions (New England BioLabs, E2621S).

Infection with UV-inactivated SINV
To generate UV-inactivated virus, SINV strain SVIA equivalent to MOI of 500 was irradiated for 7 minutes using a Stratalinker UV Crosslinker 1800 (Stratagene).Absence of infectious particles was confirmed through plaque assay on Vero cells.After UV-inactivation, HeLa cells were exposed to virus particles for 1 h and then washed three times to remove extracellular or loosely bound virus before fresh culture media was added.Lysates were harvested for western blot analysis at timed intervals to chase the clearance of capsid protein.To block autophagy induction and flux, 5 µM of PIK-III inhibitor (Selleck Chemicals, S7683), 100nM of Bafilomycin A1 (Sigma-Aldrich B1793), or 50nM of Epoxomicin (Selleck Chemicals, S7038) were added 30 minutes after cells were exposed to UV-inactivated SINV.

Fluorescence microscopy
For all cell culture microscopy experiments, HeLa cells and cortical neurons were cultured on Nunc Lab-Tek II 4 or 8-well glass chamber slides (Thermo Fisher Scientific).After exposure to autophagy inducing conditions, cells were fixed for 7 minutes with 4% PFA in PBS at room temperature and washed three times with PBS.For detection of GFP-LC3 or mCherry-capsid, a coverslip was mounted onto each slide with VECTASHIELD Antifade Mounting Medium with DAPI (Vector Laboratories H-1200), the perimeter of the coverslip was sealed with nail polish and allowed to dry overnight at room temperature before imaging.For immunofluorescence, both HeLa cells and cortical neurons were permeabilized after PFA fixation with 100% methanol chilled at -20℃ for 20 minutes followed by 1 h blocking with PBS containing 3% BSA (Sigma-Aldrich; A9418).Cells were stained with antibodies against SIN capsid, SQSTMI/p62 and phospho-SQSTM1/p62 for 2 h at room temperature in blocking buffer.After three washes, cells were treated with Alexa Fluor secondary antibodies for 1 h at room temperature, washed three times, and then mounted.All fluorescence imaging was performed using a Zeiss AxioImager Z2 microscope equipped with a Photometrics CoolSnap HQ2 CCD camera using a Zeiss PLAN APOCHROMAT 20X/0.8NA wide-field objective or PLAN APOCHROMAT 40X/0.9NA oil immersion objective and a Nikon CSU-W1 SoRa spinning disk confocal microscope.In all experiments a secondary antibody control was used for thresholding the background.Images were taken randomly of areas with similar cell density with the experimenter blinded to the experimental condition.HeLa cells SINV capsid/p62 and phospho-p62/total p62 images were taken in 0.3um Zstacks and deconvoluted with AutoQuant X3 software.Image analysis of the p62 inclusion bodies was performed using Fiji software through a custom-written macro.LAMP1 and capsid colocalization analysis was performed through Imaris software.

Transmission electron microscopy
CDKL5 WT and KO HeLa cells were infected with HSV-1 ΔBBD strain at an MOI of 10 for 12 hours.Cells were fixed for 10 minutes with 2.5% glutaraldehyde + 2% Paraformaldehyde in 0.1M cacodylate buffer.Fixed samples were washed in 0.1 M sodium cacodylate buffer and treated with 0.1% Millipore-filtered cacodylate buffered tannic acid, postfixed with 1% buffered osmium tetroxide, and stained en bloc with 1% Millipore-filtered uranyl acetate.The samples were dehydrated in increasing concentrations of ethanol, infiltrated, and embedded in LX-112 medium.The samples were polymerized in a 60°C oven for approximately 3 days.Ultrathin sections were cut in a Leica Ultracut microtome (Leica, Deerfield, IL), stained with uranyl acetate and lead citrate and examined in a JEM 1010 transmission electron microscope (JEOL, USA, Inc., Peabody, MA) at an accelerating voltage of 80 kV.Digital images were obtained using AMT Imaging System (Advanced Microscopy Techniques Corp, Danvers, MA).

SINV growth curves
To perform high MOI or low MOI multi-step viral growth curve analysis, HeLa cells were cultured in triplicate on 6-well plates and infected with either MOI of 10 or 0.01, respectively for 1.5 h.Virus infection media was replaced with fresh culture media.One hundred microliter samples were drawn from each well at specified time intervals and virus titers determined through Vero cell plaque assays.

Cell death assay
For measurement of virus-induced cell death, cells were mock-infected or infected with SINV (MOI 10) for 24 h and then processed using the CellTiter-Glo Luminescent Cell Viability Assay, per manufacturer's instructions (Promega, G7570).Luminescence was detected using a CLARIOstar Plate Reader (BMG Labtech).

Co-immunoprecipitation
To investigate the interactions between CDKL5, p62 and SINV capsid, 10 7 HeLa cells were cultured on 150 mm plates and either mock-infected or infected for 7 h at MOI of 10 with recombinant SINV expressing HA-capsid.HeLa cells were scrapped from the plate and lysed in ice-cold lysis buffer (50 mM Tris-HCL (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100) containing complete proteinase inhibitor cocktail (Roche) and Halt phosphatase inhibitor cocktail (Thermo Fisher Scientific) for 30 min on ice.Cellular debris was removed with centrifugation for 10 minutes at 15,000 g at 4℃. Supernatants were pre-cleared with either dynabeads protein G (Thermo Fisher; 10003D) or protein G PLUS agarose beads (SCBT, sc-2002) for 1.5h at 4℃ with gentle agitation.After pelleting the beads, supernatants were mixed with either 0.8 µg/mL of rat anti HA antibody (Sigma-Aldrich, 11867423001) and 30 µL of pre-washed dynabeads protein G (Thermo Fisher; 10003D) or 40ul of pre-washed anti-Flag M2 affinity gel (Millipore Sigma; A2220) and then incubated at 4℃ overnight with gentle agitation.Dynabeads were pelleted using the DynaMag-2 Magnet (Thermo Fisher, 12321D) and anti-Flag beads by centrifugation at 3000g for 1 minute followed by 3 washes with ice-cold lysis buffer and two additional washes with lysis buffer containing 300mM NaCl.Immunoprecipitated proteins were eluted by adding 2X Laemmli sample buffer (Bio-Rad Laboratories) containing 5% β-mercaptoethanol (Bio-Rad Laboratories) and boiling sample for 7 minutes followed by western blot analysis.

Affinity purification of ubiquitinated capsid using TUBEs
To determine whether SINV capsid is ubiquitinated during HeLa cells infection, we used magnetic bead fused TUBEs (LifeSensors Inc; UM401M) according to manufactures instructions.
Briefly, cell lysates were mixed with 50µL of TUBE or control magnetic beads (LifeSensors Inc; UM500M) and incubated at 4°C for 2 hours.The TUBE and control beads were then washed five times with TBST, resuspended in 2x Laemmli sample buffer, boiled and subjected to SDS-PAGE.

Protein lysate preparation and Western blot analyses
HeLa cells and cortical neurons were lysed with ice-cold 1X RIPA buffer (Cell Signaling Technology, 9806) supplemented with proteinase inhibitor cocktail (Sigma) and Halt phosphatase inhibitor cocktail (Thermo Fisher Scientific) and incubated on ice for 30 min.The insoluble components of the lysates were pelleted by centrifugation at 15,000 g at 4℃ for 15 min, and the residual supernatants mixed with 2X Laemmli sample buffer containing 5% β-mercaptoethanol.
For analysis of p62 in total lysates, cells were directly lysed in 2x Laemmli sample buffer, boiled for 3 minutes in a 100℃ heating block, and then sonicated in an ultrasonic bath for one minute.
For protein analysis in the soluble versus insoluble pellet, the pellet was washed with 1X RIPA buffer and resuspended with 2X Laemmli sample buffer at a ratio of 3:1.Nuclear/cytoplasmic fractionations were performed as previously described (5).Proteins were separated on gradient 4-20% Mini-PROTEAN TGX precast protein gels (Bio-Rad Laboratories) and transferred onto PVDF membranes (Bio-Rad Laboratories).For blocking, membranes were incubated in 5% nonfat milk in either 1X TBS or PBS containing 0.01% Tween 20.Primary and secondary antibodies were diluted in blocking buffer.Proteins were detected with SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific) using a Bio-Rad Chemidoc imager.Band densitometry was determined through Fiji software.

Flow Cytometry
WT and CDKL5 KO HeLa cells infected with SINV/mCherry-capsid were dissociated with 0.25% trypsin, fixed with 4% PFA for 5 minutes and resuspended in PBS with 3% BSA.Cells positive for mCherry-Capsid were detected through an LSRII-HTS flow cytometer (BD Biosciences) and data analyzed using FlowJo software (Version 9).

In vitro Kinase Assays
For the reaction, 0.3 µg of recombinant human CDKL5 (1-498) (ThermoFisher, A33353) was combined with 3 µg of recombinant human SQSTM1/p62 protein (GeneTex, GTX68012-pro) in buffer containing 20 mM HEPES (pH 7.5) and 10 mM MgCl2 in the presence or absence of 50 µM [γ-32 P] ATP.The reaction was incubated at 30℃ for up to 45 minutes, stopped by adding 4x Laemmli sample buffer, then subjected to SDS-PAGE for Coomassie staining and radioactivity detection on x-ray film.This same reaction was also done using recombinant CDKL5 or TBK-1 (positive control) combined with recombinant p62 protein, with or without 100 µM nonradioactive ATP, and phosphorylation of p62 detected by western blot using phosphospecific antibodies.To compare the ability of WT and KD CDKL5 to phosphorylate recombinant p62 protein at Thr269/Ser272, HeLa cells stably expressing EV, WT or KD CDKL5 were lysed in icecold buffer (50 mM HEPES (pH 7.5), 300 mM NaCl, 1% Triton X-100) supplemented with proteinase inhibitor cocktail (Sigma) and Halt phosphatase inhibitor cocktail (Thermo Fisher Scientific).Pull down of FLAG was performed as described above with anti-Flag M2 affinity gel.
The beads were washed five times with lysis buffer containing 0.5 M NaCl and then three times with the in vitro kinase buffer (20 mM HEPES (pH 7.5), 10 mM MgCl2, and 1 mM DTT) before resuspending the beads in 20 µl of in vitro kinase buffer with 100 µM ATP.The reaction was incubated at 30℃ for 45 minutes.Phosphorylation analysis was performed using Western blot.

Virus infection of mice
Mice were inoculated intracerebrally into the right cerebral hemisphere with SINV and HSV-1 and subcutaneously with CHIKV.Viruses were diluted in Hank's Balanced Salt Solution (HBSS, Thermo Fisher Scientific) and 30μL inoculum was used per mouse.For SINV, the dsTE12Q strain was used to infect seven-day-old CDKL5 WT and CDKL5 KO neonates with 1x10 3 pfu.Separately, seven-day-old neonates were infected with 1x10 5 pfu of CHIKV strain 06-02.For HSV-1 infection, 5x10 4 pfu of HSV-1ΔBBD strain was used to infect anesthetized eight-to tenweek-old littermate CDKL5 WT and CDKL5 KO mice.For mortality studies, mice were monitored daily for 21 days.To perform brain tissue analysis in SINV infected mice, mice from each genotype were randomly selected for dissection at days 1, 4 and 7.The right hemisphere was snap frozen in liquid nitrogen, homogenized in HBSS and used for plaque assay titration to determine the viral load.Brain homogenates from SINV infected mice were also assessed for IFN-b levels through mouse IFN-b ELISAs per manufacturer's instructions (Abcam, ab252363).The left hemisphere was fixed in 4% PFA, cryoprotected in 30% sucrose and embedded in paraffin for histology studies.